CN117617110A - Improvement method of dominant genic male sterile homozygous clone of brassica napus - Google Patents
Improvement method of dominant genic male sterile homozygous clone of brassica napus Download PDFInfo
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- CN117617110A CN117617110A CN202311596272.8A CN202311596272A CN117617110A CN 117617110 A CN117617110 A CN 117617110A CN 202311596272 A CN202311596272 A CN 202311596272A CN 117617110 A CN117617110 A CN 117617110A
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 240000002791 Brassica napus Species 0.000 title claims description 8
- 235000011293 Brassica napus Nutrition 0.000 title claims description 8
- 230000006872 improvement Effects 0.000 title description 3
- 241000196324 Embryophyta Species 0.000 claims abstract description 44
- 230000000306 recurrent effect Effects 0.000 claims abstract description 43
- 239000003147 molecular marker Substances 0.000 claims abstract description 13
- 230000008186 parthenogenesis Effects 0.000 claims abstract description 10
- 240000007124 Brassica oleracea Species 0.000 claims abstract description 8
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims abstract description 8
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims abstract description 8
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims abstract description 8
- 238000009396 hybridization Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 6
- 230000002068 genetic effect Effects 0.000 claims abstract description 3
- 238000009395 breeding Methods 0.000 claims description 13
- 230000001488 breeding effect Effects 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 6
- 230000035558 fertility Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 230000035800 maturation Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 230000009466 transformation Effects 0.000 description 8
- 241000231392 Gymnosiphon Species 0.000 description 6
- 230000008901 benefit Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000009405 line breeding Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/022—Genic fertility modification, e.g. apomixis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/022—Genic fertility modification, e.g. apomixis
- A01H1/024—Female sterility
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for improving a homozygous sterile line of a dominant genic male sterile line of cabbage type rape, which is based on the genetic characteristics of the dominant genic male sterile line of the cabbage type rape, uses the sterile plant (genotype Ms 5) b Ms5 c ) Is a female parent, a good variety (genotype Ms5 c Ms5 c Recurrent parent) is used as a male parent, and a heterozygous two-type line of the gene close to the recurrent parent is cultivated through hybridization and continuous backcross; then directly to the cultivated heterozygous intragenic sterile strain (genotype Ms 5) b Ms5 c ) Bagging to induce parthenogenesis to establish a clone; finally, screening out homozygous sterile clone (genotype Ms 5) of the recurrent parent near isogenic by molecular marker assisted selection b Ms5 b ) The workload of backcrossing and the like is reduced.
Description
Technical Field
The invention relates to the technical field of cabbage type rape breeding, in particular to an improvement method of a dominant genic male sterile homozygous clone of cabbage type rape.
Background
Cabbage type rape as world-wide-important oil crop, its hybridThe advantage is that the yield and the quality of the plant are improved, and the stress resistance of the plant is enhanced. The seed production method of the dominant genic male sterile three-line of the cabbage type rape is based on multiple alleles (namely wild gene Ms 5) c Sterile gene Ms5 b Restoring the fertility of the sterile Gene Ms5 a The obvious and implicit relation of the three is Ms5 a >Ms5 b >Ms5 c ) The controlled genetic mode is to utilize the three lines of homozygous two-type line, temporary maintainer line and restoring line to produce rape hybrid seed. Sterile strain (genotype Ms 5) in homozygous two-type line is selected in the process of seed production b Ms5 b ) And temporary maintainer line (genotype Ms 5) c Ms5 c ) Hybridization to produce full sterile line, next year using full sterile line as female parent and restoring line (genotype Ms 5) a Ms5 a ) Hybrid seeds are produced for male parent under natural isolation condition.
Generally, in breeding, homozygous sterile lines used for breeding hybrid seeds are mainly obtained through the following steps:
(1) Heterozygous two-type line breeding of the recurrent parent near isogene: the method comprises the steps of taking a sterile plant (genotype Ms5bMs5 c) in the existing dominant genic sterile line as a female parent, taking a good variety (genotype Ms5cMs c, recurrent parent) as a male parent, and cultivating a heterozygous two-type line (genotype Ms5bMs5c+Ms5cMs5 c) of a gene close to the recurrent parent through hybridization and continuous backcrossing;
(2) And (3) breeding a restorer line of a gene close to the recurrent parent: the existing dominant genic male sterile restorer line (genotype Ms5aMs a) is used as a female parent, and the recurrent parent in (1) is used as a male parent to carry out hybridization and continuous backcross. In the backcross process, the homozygous sterile strain (genotype Ms5bMs5 b) is used as a female parent, and the backcross offspring is subjected to test cross. Backcross offspring corresponding to the separated test cross combinations are selected, and backcross is continued by recurrent parents. After 3 backcrossing, selfing and test crossing the backcrossed offspring selected strain corresponding to the test crossing combination with separated fertility, and cultivating the restoring line (genotype Ms 5) of the recurrent parent near isogene of (1) a Ms5 a )。
(3) Breeding of homozygous sterile line: the sterile strain (genotype Ms5bMs c) in the heterozygous two-type line cultivated in the step (1) is hybridized with the restorer line (genotype Ms5aMs a) cultivated in the step (2), and the strain is selected from F1 groups (genotype Ms5aMs5b+Ms5aMs c) for selfing, and the group with fertility separation of the selfing offspring is the homozygous sterile line (genotype 1 Ms5aMs5a+2Ms5aMs5b+1Ms5bMs5b).
The prior art has the following defects: the backcross transformation has large workload, namely, the genes Ms5 are needed to be used respectively a And Ms5 b Carrying out backcross transformation on the target gene; the genotyping workload is great because of the gene Ms5 a When backcross transformation is carried out for the target, the backcross offspring must be subjected to test cross identification with homozygous sterile plants to identify carrying Ms5 a Is a subject of (2); ms5 is required to be held a And Ms5 b After the same colony is fused, i.e. the backcross breeding is completed, the cultivated heterozygous two-type line and the restorer line which are close to the recurrent parent are hybridized again, and Ms5 is obtained a And Ms5 b After the same offspring group is fused, the homozygous sterile line is finally screened out through selfing, and the backcross workload is high.
Disclosure of Invention
The invention provides a method for directly improving a dominant genic male sterile homozygous asexual line of brassica napus by adopting parthenogenesis combined molecular marker assisted breeding, which effectively reduces backcross transformation and genotype identification workload, shortens breeding period and can greatly improve breeding efficiency.
An improved method for a cabbage type rape dominant genic male sterile homozygous clone comprises the following specific operation steps:
step 1.1, breeding of heterozygous two types of lines of recurrent parent near isogenic:
step 1.1.1 based on the dominant genic male sterile property of Brassica napus, the sterile plant (genotype Ms5 b Ms5 c ) Is a female parent, a good variety (genotype Ms5 c Ms5 c Recurrent parent) as male parent, F 1 The sterile plant and the fertile plant of the colony account for 50 percent respectively, and the genotypes of the sterile plant and the fertile plant are Ms5 respectively b Ms5 c And Ms5 c Ms5 c ；
Step 1.1.2 use of F 1 Genotype Ms5 in population b Ms5 c Sterile plant of (2) is used as female parent, backcrossed with recurrent parent, BC 1 The sterile plant and the fertile plant of the colony account for 50 percent respectively, and the genotypes are Ms5 respectively b Ms5 c And Ms5 c Ms5 c . Continuously backcrossing for 3 times according to the method, and cultivating a heterozygous two-type line of the recurrent parent near isogenic line;
the sterile strain and the fertile strain in the heterozygous two-type line which are approximately isogenic to the recurrent parent and are cultivated by the method respectively account for 50 percent, and the genotypes are Ms5 respectively b Ms5 c And Ms5 c Ms5 c ；
Step 1.2 obtaining homozygous clones based on parthenogenesis:
step 1.2.1, taking sterile plants in the heterozygous two-type lines which are close to the recurrent parent and are cultivated in the step 1.1.2 as materials, bagging and selfing 3-4 inflorescences at the top by using a non-woven bag in the flowering period, cutting off the main sequence or branches of the bagged plants after maturation, and collecting seeds after threshing;
step 1.2.2 the seeds collected in the step 1.2.1 are sterilized and placed in a B5 culture medium for germination, sampling is carried out according to the serial number of a single plant after the emergence of seedlings, a clone is established, and the theoretical genotype comprises Ms5 b Ms5 b And Ms5 c Ms5 c ；
Step 1.3 selection of dominant genic male sterile homozygous clone based on molecular marker assistance:
step 1.3.1 extraction of the leaf DNA taken in 1.2.2, detection of amplification by means of the molecular marker BE10, the amplification band pattern having 2 types: 398bp or 305bp amplified region, wherein the 305bp amplified region has a corresponding genotype of Ms5 b Ms5 b ；
Step 1.3.1 screening and preserving the single strain of 305bp amplified band for propagation, and obtaining the single strain (clone) which is the dominant genic male sterile homozygous clone of the method, and the genotype is Ms5 b Ms5 b 。
The invention has the following advantages:
without using Ms5 a Backcross transformation is carried out for the target gene, and genotype identification is not needed for the offspring of the backcross; without the need to map the gene Ms5 a And Ms5 b Is fused into the same group, and the homozygous sterile clone (genotype Ms 5) is directly obtained by the clone of parthenogenesis offspring b Ms5 b )；
In summary, the present invention uses the existing heterozygous sterile strain (genotype Ms5bMs c) as Ms5b donor, hybridizes and backcross with the fine variety (wild type Ms5cMs c and used as recurrent parent), and breeds the heterozygous sterile line with the fine variety near isogenic, the sterile strain rate is 50%. And the sterile plant (genotype Ms5bMs c) in the cultivated heterozygous sterile line is subjected to bagging in the flowering period, the seeds obtained by parthenogenesis are established into a clone after parthenogenesis is induced, then auxiliary selection is carried out by means of a molecular marker BE10 of a dominant genic sterile gene Ms5, and the homozygous sterile clone (genotype Ms5bMs b) of the gene close to the recurrent parent is screened out, so that the workload of backcrossing and the like is reduced.
Drawings
FIG. 1 shows the gene Ms5 of the prior art b Is a schematic diagram of a hybrid two-type line of target backcross transformation and recurrent parent near isogenes.
FIG. 2 shows the gene Ms5 of the prior art a Schematic representation of the restorer line of the target backcross transgene and recurrent parent near isogene.
FIG. 3 shows the gene Ms5 of the prior art a And Ms5 b And (3) merging the same offspring group to screen a schematic diagram of the homozygous sterile line.
FIG. 4 is a schematic representation of an improved method of the present invention for dominant genic male sterile homozygous clone of Brassica napus.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Referring to FIG. 4, an improved method for dominant genic male sterile homozygous clone of Brassica napus comprises the steps of:
step 1.1 Gene Ms5 b The breeding of heterozygous two types of lines of target backcross transfer and recurrent parent near isogenic:
step 1.1.1 Using the existing dominant genic male sterile line of the sterile plant (genotype Ms5 b Ms5 c ) Is a female parent, a good variety (genotype Ms5 c Ms5 c Recurrent parent) as male parent, F 1 The sterile plant and the fertile plant of the colony account for 50 percent respectively, and the genotypes of the sterile plant and the fertile plant are Ms5 respectively b Ms5 c And Ms5 c Ms5 c ；
Step 1.1.2F as described in 1.1.1 1 Genotype Ms5 in population b Ms5 c Sterile plant of (2) is used as female parent, backcrossed with recurrent parent, BC 1 The sterile plant and the fertile plant of the colony account for 50 percent respectively, and the genotypes are Ms5 respectively b Ms5 c And Ms5 c Ms5 c . Continuously backcrossing for 3 times according to the method, and cultivating a heterozygous two-type line of the recurrent parent near isogenic line;
the sterile strain and the fertile strain in the heterozygous two-type line which are approximately isogenic to the recurrent parent and are cultivated by the method respectively account for 50 percent, and the genotypes are Ms5 respectively b Ms5 c And Ms5 c Ms5 c ；
Step 1.2 obtaining homozygous clones based on parthenogenesis:
step 1.2.1, taking sterile plants in the heterozygous two-type lines which are cultivated in the step 1.1.2 and are close to the recurrent parent as materials, bagging and selfing 3-4 inflorescences at the top by using a non-woven bag in the flowering period, cutting off the main sequence or branches of the bagged plants after maturation, and collecting seeds after threshing;
step 1.2.2 the seeds collected in step 1.2.1 are sterilized and placed in a B5 culture medium for germination, sampling is carried out according to the serial number of a single plant after the emergence of seedlings, a clone is established, and the theoretical genotype comprises Ms5 b Ms5 b And Ms5 c Ms5 c ；
Step 1.3 selection of dominant genic male sterile homozygous clone based on molecular marker assistance:
step 1.3.1 extraction of the leaf DNA taken in 1.2.2, detection of amplification by means of the molecular marker BE10, the amplification band pattern having 2 types: 398bp or 305bp amplified region, wherein the 305bp amplified region has a corresponding genotype of Ms5 b Ms5 b ；
Step 1.3.1 screening and preserving the single strain of 305bp amplified band for propagation, and obtaining the single strain (clone) which is the dominant genic male sterile homozygous clone (genotype Ms 5) b Ms5 b )。
The invention is embodied by the present heterozygous sterile strain (genotype Ms5 b Ms5 c ) For Ms5 b Donor, and elite variety (wild-type Ms5 c Ms5 c And used as recurrent parent) and backcrossing, breeding a heterozygous sterile line which is close to the isogenic of the excellent variety, and then carrying out the anthesis on the sterile plant (genotype Ms 5) in the bred heterozygous sterile line b Ms5 c ) Bagging, establishing asexual line by using seeds obtained after parthenogenesis induction, performing auxiliary selection by means of molecular marker BE10 of dominant genic male sterile gene Ms5, and screening out homozygous sterile asexual line (genotype Ms 5) of nearly isogenic to recurrent parent b Ms5 b )。
The prior art comprises the following steps: referring to FIGS. 1-2, the gene Ms5 is targeted b And Ms5 a Performing backcross transformation to obtain heterozygous two-type line and restoring line of the recurrent parent near isogene, and performing test cross identification on the obtained restoring line of the recurrent parent near isogene and homozygous sterile strain to identify carrying Ms5 a The backcross transformation and genotyping of the individuals of (a) are heavy. Referring to fig. 3, finally, the 2 near isogenic lines obtained in fig. 1 and 2 are hybridized again and selfed, so that the homozygous sterile line can be selected, and the working life and the workload are large.
The invention has the advantages that: the existing dominant genic male sterile line is used for the inner sterile strain (genotype Ms5 b Ms5 c ) Is a female parent, a good variety (genotype Ms5 c Ms5 c Recurrent parent) is used as a male parent, and a heterozygous two-type line of the gene close to the recurrent parent is cultivated through hybridization and continuous backcross; then directly to the cultivated heterozygous intragenic sterile strain (genotype Ms 5) b Ms5 c ) Bagging to induce parthenogenesis to establish a clone; finally, screening out homozygous sterile clone (genotype Ms 5) of the recurrent parent near isogenic by molecular marker assisted selection b Ms5 b )。
The embodiment of the invention comprises the following steps:
in 2017, the full sterile line 4166CA is taken as a female parent, and the yellow seed inbred line '16-3111' bred from the double low hybrid seed 'D4818' of Guizhou province institute is taken as a male parent for hybridization.
Hybridization F in 2018 1 Sterile strain in the generation '17-3278' is used as female parent, and yellow seed inbred line '16-3111' is used as recurrent parentAnd (5) backcrossing.
BC in 2019 1 Sterile plants in the population '18-3308' are used as female parents, the yellow seed inbred line '16-3111' is used as recurrent parent, and backcross is carried out. And selecting and sowing only brown and yellow seeds after removing the black and brown seeds before sowing.
BC in 2020 2 Sterile plants in the population '19-3243' are used as female parents, the yellow seed inbred line '16-3111' is used as recurrent parent, backcross is carried out, and the seeds are brown and yellow.
BC of 2021 3 The sterile plants selected from the group '20-3225' are bagged to obtain BC 3 F 2 Brown-yellow seeds 5 grains.
BC of 2021 3 F 2 Seed '210347' is used as a donor, clone is established, and molecular marker BE10 is used for carrying out molecular marker assisted selection on 5 clone. The clones '210347-2' and '210347-5' were identified to have only 305bp amplified bands, indicating that the genotypes of these 2 clones are Ms5 b Ms5 b 。
After the clone '210347-5' is propagated, the clone is planted in a seed selection garden and is subjected to test crossing with the yellow seed inbred line '16-3111' in 2022. The fertility identification of the test cross offspring '22-3207' shows that 12 single plants are all male sterile plants.
The invention and its embodiments have been described above without limitation, and the actual construction is not limited thereto. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (2)
1. An improved method for a cabbage type rape dominant genic male sterile homozygous clone, which is characterized by comprising the following steps: the method comprises the following steps:
step 1.1, breeding of heterozygous two-type lines of the recurrent parent near isogenic:
step 1.1.1, based on the dominant genic male sterile genetic characteristic of the brassica napus, taking a sterile plant in the existing dominant genic male sterile line as a female parent, taking recurrent parent as a male parent for hybridization, and F 1 Sterile plant of colonyThe fertility strains respectively account for 50 percent, and the genotypes of the fertility strains are Ms5 respectively b Ms5 c And Ms5 c Ms5 c ；
Step 1.1.2, with Ms5 b Ms5 c Sterile plant of (2) is used as female parent, backcrossed with recurrent parent, BC 1 The sterile plant and the fertile plant of the colony account for 50 percent respectively, and the genotypes are Ms5 respectively b Ms5 c And Ms5 c Ms5 c The method comprises the steps of carrying out a first treatment on the surface of the Continuously backcrossing for 3 times according to the method, and cultivating a heterozygous two-type line of the recurrent parent near isogenic line;
step 1.2, obtaining a homozygous clone based on parthenogenesis:
step 1.2.1, taking sterile plants in the heterozygous two-type lines which are close to the recurrent parent and are cultivated in the step 1.1.2 as materials, bagging and selfing 3-4 inflorescences at the top by using a non-woven bag in the flowering period, cutting off the main sequence or branches of the bagged plants after maturation, and collecting seeds after threshing;
step 1.2.2, sterilizing the seeds, germinating in a B5 culture medium, sampling according to the serial number of a single plant after emergence of seedlings, and establishing a clone, wherein the genotype comprises Ms5 b Ms5 b And Ms5 c Ms5 c ；
Step 1.3, selecting dominant genic male sterile homozygous clone based on molecular marker assistance:
step 1.3.1, extracting the leaf DNA obtained in step 1.2.2, and carrying out amplification detection by using a molecular marker BE10, wherein the amplification band types are 2: 398bp or 305bp amplified region, wherein the 305bp amplified region has a corresponding genotype of Ms5 b Ms5 b ；
Step 1.3.1, screening and preserving the single strain of 305bp amplified band for propagation, and obtaining the dominant genic male sterile homozygous clone of the single strain, wherein the genotype is Ms5 b Ms5 b 。
2. The method for improving a dominant genic male sterile homozygous clone of brassica napus according to claim 1, wherein: the sterile plant and the fertile plant in the heterozygous two-type line which are cultivated in the step 1.1.2 and are close to isogenic with recurrent parent respectively account for 50 percent, and the genotypes are Ms5 respectively b Ms5 c And Ms5 c Ms5 c 。
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